Summary Brain microvessels play an important role in the neurovascular coupling (NVC). Mitochondria are energy sensors of cells. My work for the first time demonstrated the link between mitochondrial depolarization and activation of nitric oxide synthases (NOS). Recently, we have made a novel discovery of a neuronal NOS (nNOS) isoform in endothelial cells that uniquely produce reactive oxygen species (ROS). The nNOS is co-expressed with NO- producing eNOS in endothelial cells and both isoforms are involved in the bidirectional regulation of mitochondria. Diabetes mellitus (DM) increases the risk of cerebrovascular dysfunction and dementia. Importantly, hypoglycemia is a dangerous side effect of DM treatments, particularly insulin-therapy. Patients with DM often experience mild hypoglycemia, but these episodes are unaccounted for in determining the cardiovascular morbidity and mortality. We achieved a technological breakthrough utilizing Seahorse XFe24 analyzer and determined the mitochondrial respiration and cellular bioenergetics of brain microvessels. We made a novel observation that five episodes of recurrent hypoglycemia (RH) impaired the microvascular mitochondrial function. Notably, single episode of acute mild or severe hypoglycemia as well as Impairments of NOS activity was found to mediate RH-induced alterations of cellular bioenergetics. Thus, we hypothesize that mild RH disrupts NVC by promoting microvascular mitochondrial dysfunction leading to impaired cognitive function. We further hypothesize that increased nNOS-induced oxidative stress coupled with reduced eNOS-derived NO contribute to the mitochondrial dysfunction following RH. We propose to use streptozotocin treated C57Bl/6 mice and db/db mice with leptin receptor mutation as models of diabetes with untreated mice as controls. In addition, we will employ eNOS knockout and inducible endothelial cell specific nNOS knockout mice to investigate the role of NOS isoforms in RH-induced microvascular dysfunction. Each animal will be subjected to five episodes (one per day) of mild (blood glucose 70-80 mg/dl) or severe (blood glucose 40-54 mg/dl) insulin-induced hypoglycemia or saline control. Aim 1 is to demonstrate that mild and severe RH (in vivo) can increase the production of NOS-derived ROS and display RH-induced functional mitochondrial respiration deficits in cerebral microvessels (ex vivo). Aim 2 is to establish the impact of RH on NVC in vivo. We will determine the RH-induced deficits in NVC by measuring the changes in arteriolar and capillary diameter in response to neuronal activation (whisker stimulation) in awake mice using two-photon laser scanning microscopy. Aim 3 is to determine the impact of RH on cognitive function using novel texture discrimination task and modified Y-maze test. The results of this proposal would identify the mechanistic link between mild RH and the cerebral microvascular mitochondria dysfunction and challenge the existing dogma to demonstrate that mild RH is equally detrimental as severe RH in contributing to the DM-induced cerebrovascular dysfunction, impaired NV, and cognitive dysfunction.